Development of a Novel Anti-CD44 Variant 6 Monoclonal Antibody C44Mab-9 for Multiple Applications against Colorectal Carcinomas

CD44 is a cell surface glycoprotein, and its isoforms are produced by the alternative splicing with the standard and variant exons. The CD44 variant exon-containing isoforms (CD44v) are overexpressed in carcinomas. CD44v6 is one of the CD44v, and its overexpression predicts poor prognosis in colorectal cancer (CRC) patients. CD44v6 plays critical roles in CRC adhesion, proliferation, stemness, invasiveness, and chemoresistance. Therefore, CD44v6 is a promising target for cancer diagnosis and therapy for CRC. In this study, we established anti-CD44 monoclonal antibodies (mAbs) by immunizing mice with CD44v3-10-overexpressed Chinese hamster ovary (CHO)-K1 cells. We then characterized them using enzyme-linked immunosorbent assay, flow cytometry, western blotting, and immunohistochemistry. One of the established clones (C44Mab-9; IgG1, kappa) reacted with a peptide of the variant 6-encoded region, indicating that C44Mab-9 recognizes CD44v6. Furthermore, C44Mab-9 reacted with CHO/CD44v3-10 cells or CRC cell lines (COLO201 and COLO205) by flow cytometry. The apparent dissociation constant (KD) of C44Mab-9 for CHO/CD44v3-10, COLO201, and COLO205 was 8.1 × 10−9 M, 1.7 × 10−8 M, and 2.3 × 10−8 M, respectively. C44Mab-9 detected the CD44v3-10 in western blotting, and partially stained the formalin-fixed paraffin-embedded CRC tissues in immunohistochemistry. Collectively, C44Mab-9 is useful for detecting CD44v6 in various applications.


Introduction
Colorectal cancer (CRC) has become the second cancer type for the estimated deaths in men and women combined in the United States, 2023 [1]. The development of CRC is classically explained by Fearon and Vogelstein model; the sequential genetic changes including APC (adenomatous polyposis coli), KRAS, DCC (deleted in colorectal cancer, chromosome 18q), and TP53 lead to CRC progression [2]. However, CRC exhibits heterogeneous outcomes and drug responses. Therefore, the large-scale data analysis by an international consortium classified the CRC into four consensus molecular subtypes, including the microsatellite instability immune, the canonical, the metabolic, and the mesenchymal types [3]. In addition, various marker proteins have been investigated for the prediction of prognosis and drug responses of CRC [4,5]. Among them, recent studies suggest that CD44 plays a critical role in tumor progression through its cancer-initiating and metastasis-promoting properties [6].
CD44 is a polymorphic integral membrane protein, which binds to hyaluronic acid (HA), and contributes to cell-matrix adhesion, cell proliferation, migration, and tumor metastasis [7]. When the CD44 is transcribed, its pre-messenger RNA can be received alternative splicing and maturated into mRNAs that encode various CD44 isoforms [8]. The mRNA assembles with ten standard exons and the sixth variant exon encodes CD44v6, which plays critical roles in cell proliferation, migration, survival, and angiogenesis [9,10]. Functionally, CD44v6 can interact with HA via the standard exons-encoded region [11]. Furthermore, the v6-encoded region functions as a co-receptor of various receptors for epidermal growth factor, hepatocyte growth factor, C-X-C motif chemokine 12, and osteopontin [12]. Therefore, the receptor tyrosine kinase or G protein-coupled receptor signaling pathways are potentiated in the presence of CD44v6 [13]. These functions are essential for homeostasis or regeneration in normal tissues. Importantly, CD44v6 overexpression plays a critical role in CRC progression. For instance, CD44v6 is involved in colorectal carcinoma invasiveness, colonization, and metastasis [14]. Therefore, CD44v6 is a promising target for cancer diagnosis and therapy.
The clinical significance of CD44v6 in CRC deserves consideration. Anti-CD44v6 therapies mainly include the blocking of the v6-encoded region by monoclonal antibody (mAb) [12]. First, humanized anti-CD44v6 mAbs (BIWA-4 and BIWA-8) labeled with 186 Re exhibited therapeutic efficacy in head and neck squamous cell carcinoma (SCC) xenograft-bearing mice [15]. Furthermore, the humanized anti-CD44v6 mAb, bivatuzumabmertansine (anti-tubulin agent) conjugate, was evaluated in clinical trials [16]. However, the clinical trials were discontinued due to severe skin toxicity, including a case of lethal epidermal necrolysis [17]. The efficient accumulation of mertansine was most likely responsible for the high toxicity [17,18]. Therefore, the development of anti-CD44v6 mAbs with more potent and fewer side effects is desired.
We also examined the antitumor effects of C 44 Mab-5 in mouse xenograft models [24]. We converted the mouse IgG 1 subclass antibody (C 44 Mab-5) into an IgG 2a subclass antibody (5-mG 2a ), and further produced a defucosylated version (5-mG 2a -f) using FUT8-deficient ExpiCHO-S (BINDS-09) cells. In vitro analysis demonstrated that 5-mG 2a -f showed moderate antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity activities against HSC-2 and SAS oral cancer cells. In vivo analysis revealed that 5-mG 2a -f significantly reduced tumor growth in HSC-2 and SAS xenografts in comparison to control mouse IgG, even after injection seven days post-tumor inoculation. These results suggested that treatment with 5-mG 2a -f may represent a useful therapy for patients with CD44-expressing oral cancers.
For epitope mapping of C 44 Mab-5, we employed the RIEDL tag system ("RIEDL" peptide and LpMab-7 mAb) [23]. We inserted the "RIEDL" peptide into the CD44 protein from the 21st to 41st amino acids. The transfectants produced were stained by LpMab-7 and C 44 Mab-5 in flow cytometry. C 44 Mab-5 did not react with the 30th-36th amino acids of the deletion mutant of CD44. Further, the reaction of C 44 Mab-5 to RIEDL tag-inserted CD44 from the 25th to 36th amino acids was lost, although LpMab-7 detected most of the RIEDL tag-inserted CD44 from the 21st to 41st amino acids. These results indicated that the epitope of C 44 Mab-5 for CD44 was determined to be the peptide from the 25th to 36th amino acids of CD44 using the RIEDL insertion for epitope mapping (REMAP) method.
In this study, we developed a novel anti-CD44v6 mAb, C 44 Mab-9 (IgG 1 , kappa) by CBIS method, and evaluated its applications, including flow cytometry, western blotting, and immunohistochemical analyses.

Results
2.1. Establishment of Anti-CD44v6 mAb, C 44 Mab-9 We employed the CBIS method to develop anti-CD44 mAbs. In the CBIS method, we prepared a stable transfectant as an immunogen. Then, we performed the high throughput hybridoma screening using flow cytometry ( Figure 1). In this study, mice were immunized with CHO/CD44v3-10 cells. Hybridomas were seeded into 96-well plates, and CHO/CD44v3-10-positive and CHO-K1-negative wells were selected. After limiting dilution, anti-CD44 mAb-producing clones were finally established. We next performed an enzyme-linked immunosorbent assay (ELISA) to determine the epitope of each mAb. Among them, C 44 Mab-9 (IgG 1 , kappa) was shown to recognize the only CD44p351-370 peptide (EETATQKEQWFGNRWHEGYR), which is corresponding to variant 6-encoded sequence ( Table 1). RIEDL tag-inserted CD44 from the 21st to 41st amino acids. These results indicated that the epitope of C44Mab-5 for CD44 was determined to be the peptide from the 25th to 36th amino acids of CD44 using the RIEDL insertion for epitope mapping (REMAP) method.
In this study, we developed a novel anti-CD44v6 mAb, C44Mab-9 (IgG1, kappa) by CBIS method, and evaluated its applications, including flow cytometry, western blotting, and immunohistochemical analyses.

Establishment of Anti-CD44v6 mAb, C44Mab-9
We employed the CBIS method to develop anti-CD44 mAbs. In the CBIS method, we prepared a stable transfectant as an immunogen. Then, we performed the high throughput hybridoma screening using flow cytometry ( Figure 1). In this study, mice were immunized with CHO/CD44v3-10 cells. Hybridomas were seeded into 96-well plates, and CHO/CD44v3-10-positive and CHO-K1-negative wells were selected. After limiting dilution, anti-CD44 mAb-producing clones were finally established. We next performed an enzyme-linked immunosorbent assay (ELISA) to determine the epitope of each mAb. Among them, C44Mab-9 (IgG1, kappa) was shown to recognize the only CD44p351-370 peptide (EETATQKEQWFGNRWHEGYR), which is corresponding to variant 6-encoded sequence ( Table 1).

Figure 1.
A schematic illustration of ant-human CD44 mAbs production. A BALB/c mouse was intraperitoneally immunized with CHO/CD44v3-10 cells. Hybridomas were produced by the fusion of the splenocytes and P3U1 cells. Then, the screening was performed by flow cytometry using parental CHO-K1 and CHO/CD44v3-10 cells. After cloning and additional screening, a clone C44Mab-9 (IgG1, kappa) was established. Finally, the binding epitopes were determined by enzymelinked immunosorbent assay (ELISA) using peptides that cover the extracellular domain of CD44v3-10. Figure 1. A schematic illustration of ant-human CD44 mAbs production. A BALB/c mouse was intraperitoneally immunized with CHO/CD44v3-10 cells. Hybridomas were produced by the fusion of the splenocytes and P3U1 cells. Then, the screening was performed by flow cytometry using parental CHO-K1 and CHO/CD44v3-10 cells. After cloning and additional screening, a clone C 44 Mab-9 (IgG 1 , kappa) was established. Finally, the binding epitopes were determined by enzyme-linked immunosorbent assay (ELISA) using peptides that cover the extracellular domain of CD44v3-10.

Western Blot Analysis
We next performed western blot analysis to assess the sensitivity of C44Mab-9. To

Immunohistochemical Analysis Using C44Mab-9 against Tumor Tissues
We next examined whether C44Mab-9 could be used for immunohistochemical an yses using formalin-fixed paraffin-embedded (FFPE) sections. Since previous an CD44v6 mAbs could detect CD44v6 in SCC tissues at a high frequency, we first stain an oral SCC tissue. As shown in Figure 5A, C44Mab-9 exhibited clear membranous sta ing, and could clearly distinguish tumor cells from stromal tissues. In contrast, C44Mab stained both ( Figure 5B). We next investigated CRC sections. C44Mab-9 showed memb nous staining in CRC cells, but not stromal tissues ( Figure 5C). In contrast, C44Mab-46 a stained both ( Figure 5D). These results indicated that C44Mab-9 is useful for immunohis chemical analysis of FFPE tumor sections.

Immunohistochemical Analysis Using C 44 Mab-9 against Tumor Tissues
We next examined whether C 44 Mab-9 could be used for immunohistochemical analyses using formalin-fixed paraffin-embedded (FFPE) sections. Since previous anti-CD44v6 mAbs could detect CD44v6 in SCC tissues at a high frequency, we first stained an oral SCC tissue. As shown in Figure 5A, C 44 Mab-9 exhibited clear membranous staining, and could clearly distinguish tumor cells from stromal tissues. In contrast, C 44 Mab-46 stained both ( Figure 5B). We next investigated CRC sections. C 44 Mab-9 showed membranous staining in CRC cells, but not stromal tissues ( Figure 5C). In contrast, C 44 Mab-46 also stained both ( Figure 5D). These results indicated that C 44

Discussion
In this study, we developed C44Mab-9 using the CBIS method (Figure 1), and determined its epitope as variant 6 encoded region (Table 1). Then, we showed the usefulness of C44Mab-9 for multiple applications, including flow cytometry (Figures 2 and 3), western blotting (Figure 4), and immunohistochemistry ( Figure 5).
Anti-CD44v6 mAbs (clones 2F10, VFF4, VFF7, and VFF18) were previously developed, and mainly used for tumor diagnosis and therapy. The 2F10 was established by the immunization of CD44v3-10-Fc protein produced by COS1 cells. The exon specificity of the 2F10 was determined by indirect immunofluorescent staining of COS1 cells transfected with human CD44v cDNAs, including CD44v3-10, CD44v6-10, CD44v7-10, CD44v8-10, and CD44v10 [25]. Therefore, the 2F10 is thought to recognize the peptide or glycopeptide structure of CD44v6. However, the detailed binding epitope of 2F10 has not been determined.

Discussion
In this study, we developed C 44 Mab-9 using the CBIS method (Figure 1), and determined its epitope as variant 6 encoded region (Table 1). Then, we showed the usefulness of C 44 Mab-9 for multiple applications, including flow cytometry (Figures 2 and 3), western blotting (Figure 4), and immunohistochemistry ( Figure 5).
Anti-CD44v6 mAbs (clones 2F10, VFF4, VFF7, and VFF18) were previously developed, and mainly used for tumor diagnosis and therapy. The 2F10 was established by the immunization of CD44v3-10-Fc protein produced by COS1 cells. The exon specificity of the 2F10 was determined by indirect immunofluorescent staining of COS1 cells transfected with human CD44v cDNAs, including CD44v3-10, CD44v6-10, CD44v7-10, CD44v8-10, and CD44v10 [25]. Therefore, the 2F10 is thought to recognize the peptide or glycopeptide structure of CD44v6. However, the detailed binding epitope of 2F10 has not been determined.
The VFF series mAbs were established by the immunization of bacterial-expressed CD44v3-10 fused with glutathione S-transferase [26,27]. Afterward, VFF4 and VFF 7 were used in the immunohistochemical analysis [28], and VFF18 was humanized as BIWA-4 [15], and developed to bivatuzumab-mertansine drug conjugate for clinical trials [17,18]. The VFF18 bound only to the fusion proteins, containing a variant 6-encoded region. Furthermore, the VFF18 recognized several synthetic peptides, spanning the variant 6encoded region in ELISA, and the WFGNRWHEGYR peptide was determined as the epitope [26]. As shown in Table 1, C 44 Mab-9 also recognized a synthetic peptide (CD44p351-370), which possesses the above sequence. In contrast, a synthetic peptide (CD44p361-380) possesses the FGNRWHEGYR sequence, which is not recognized by C 44 Mab-9. Therefore, C 44 Mab-9 and VFF18 recognize CD44v6 with a similar variant 6-encoded region. Detailed epitope mapping for C 44 Mab-9 is required in the future.
A mutated version of BIWA-4, called BIWA-8, was constructed for improving binding affinity. This was achieved by two amino acid mutations of the light chain without changing the humanized heavy chain [15]. The BIWA-8 was further engineered to chimeric antigen receptors (CARs). The CD44v6 CAR-T exhibited antitumor effects against primary human acute myeloid leukemia and multiple myeloma cells in immunocompromised mice [29]. Furthermore, the CD44v6 CAR-T also showed efficacy in xenograft models of lung and ovarian carcinomas [30], which is expected for a wider development toward solid tumors. However, Greco et al. demonstrated that the N-glycosylation of CD44v6 protects tumor cells from the CD44v6 CAR-T targeting [31]. This phenomenon is probably due to the masking of CD44v6 CAR binding by the N-glycosylation because the original VFF18 was established by bacterial-expressed CD44v3-10 immunization and recognized the peptidic epitope lacking the N-glycosylation [26]. In contrast, C 44 Mab-9 was established by immunization of CHO/CD44v3-10 cells, but recognizes a synthetic peptide (Table 1). Meanwhile, C 44 Mab-9 could detect more than 180 kDa, heavily glycosylated CD44v3-10 in western blot analysis ( Figure 4). Further studies are required to reveal whether the N-glycosylation affects the recognition by C 44 Mab-9 for future application to CAR-T therapy.
The clinical significance of CD44v6 expression in patients with CRC using immunohistochemical analysis remains controversial. The elevated expression has been associated with poor prognosis, linked to adverse prognosis [32,33]. However, others have reported that CD44v6 expression is associated with a favorable outcome [34,35]. Various clones of anti-CD44v6 mAbs appeared to influence the outcome of the clinical significance. Among these clinical studies, Saito et al. used VFF18 and showed similar staining patterns of C 44 Mab-9 ( Figure 5). They also found that CD44v6 expression was observed in poorly differentiated CRC without E-cadherin expression. Furthermore, the high CD44v6 expression exhibited a significant inverse correlation with E-cadherin expression and was found to be an independent poor prognostic factor in disease-free survival and overall survival [36]. In the future, we should evaluate the clinical significance of the C 44 Mab-9-positive CRC with E-cadherin expression.
Large-scale genomic analyses of CRCs defined 4 subtypes: (1) microsatellite instability immune; (2) canonical; (3) metabolic; (4) mesenchymal types [3]. Since the CD44v6 expression was observed in a part of CRC tissues (Figure 5), the relationship to the subtypes should be evaluated. In addition, the mechanism of CD44v6 upregulation including the transcriptional regulation and the v6 inclusion by alternative splicing should be determined. The inclusion of CD44 variant exons was reported to be promoted by the ERK-Sam68 axis [37]. Moreover, CD44v6 forms a ternary complex with MET and HGF, which is essential for the c-MET activation [38]. This positive feedback is a potential mechanism to promote the variant exon inclusion.
CD44v6-positive CRC cells exhibited cancer-initiating cell properties [39]. Cytokines, HGF, C-X-C motif chemokine 12, and osteopontin, secreted from tumor-associated fibroblasts, promote the CD44v6 expression in the cancer-initiating cells, which promotes migration and metastasis of CRC cells [14]. Clinically, circulating-tumor cells (CTCs), which express EpCAM, MET, and CD44, identify a subset with increased metastasis-initiating phenotype [40], suggesting that CD44v6 plays an important role in cancer-initiating cell property cooperating with MET. In addition, CTC culture methods, including two-dimensional (2D) expansion, 3D organoids/spheroids culture, and xenograft formation in mice, have been developed to evaluate the character of CTCs [41]. Therefore, the biological property to affect cell proliferation and invasiveness by C 44 Mab-9 should be investigated because CD44v6 can potentiate the MET signaling by forming the ternary complex with HGF [38]. Therefore, it would be valuable to examine the effect of C 44 Mab-9 on CTC proliferation in vitro and metastasis in vivo.
To evaluate the in vivo effect, we previously converted the IgG 1 subclass of mAbs into a mouse IgG 2a , and produced a defucosylated version. These defucosylated IgG 2a mAbs exhibited potent ADCC in vitro, and reduced tumor growth in mouse xenograft models [24,[42][43][44][45][46][47][48]. Therefore, the production of a class-switched and defucosylated version of C 44 Mab-9 is required to evaluate the antitumor activity in vivo.

Hybridoma Production
The female BALB/c mice (6-weeks old) were purchased from CLEA Japan (Tokyo, Japan). Animals were housed under specific pathogen-free conditions. All animal experiments were also conducted according to relevant guidelines and regulations to minimize animal suffering and distress in the laboratory. The Animal Care and Use Committee of Tohoku University (Permit number: 2019NiA-001) approved animal experiments. The mice were monitored daily for health during the full four-week duration of the experiment. A reduction of more than 25% of the total body weight was defined as a humane endpoint. During sacrifice, the mice were euthanized through cervical dislocation, after which death was verified through respiratory and cardiac arrest. The mice were intraperitoneally immunized with CHO/CD44v3-10 (1 × 10 8 cells) and Imject Alum (Thermo Fisher Scientific Inc.) as an adjuvant, which stimulates a nonspecific immune response for mixed antigens using this formulation of aluminum hydroxide and magnesium hydroxide. After three additional immunizations of CHO/CD44v3-10 (1 × 10 8 cells), a booster injection of CHO/CD44v3-10 was intraperitoneally administered 2 days before harvesting the spleen cells. The splenocytes were fused with P3U1 cells using polyethylene glycol 1500 (PEG1500; Roche Diagnostics, Indianapolis, IN, USA). The supernatants, which are positive for CHO/CD44v3-10 cells and negative for CHO-K1 cells, were selected by the flow cytometry-based high throughput screening using SA3800 Cell Analyzers (Sony Corp., Tokyo, Japan).

Determination of Dissociation Constant (K D ) by Flow Cytometry
Serially diluted C 44 Mab-9 was suspended with CHO/CD44v3-10, COLO201, and COLO205 cells. The cells were further treated with Alexa Fluor 488-conjugated anti-mouse IgG (1:200). Fluorescence data were collected using BD FACSLyric and analyzed using BD FACSuite software version 1.3 (BD Biosciences). To determine the dissociation constant (K D ), GraphPad Prism 8 (the fitting binding isotherms to built-in one-site binding models; GraphPad Software, Inc., La Jolla, CA, USA) was used.

Immunohistochemical Analysis
The FFPE oral SCC tissue was obtained from Tokyo Medical and Dental University [69]. FFPE sections of colorectal carcinoma tissue array (Catalog number: CO483a) were purchased from US Biomax Inc. (Rockville, MD, USA). The sections were autoclaved in citrate buffer (pH 6.0; Nichirei biosciences, Inc., Tokyo, Japan) for 20 min. After blocking with SuperBlock T20 (Thermo Fisher Scientific, Inc.), the sections were incubated with C 44 Mab-9 (1 µg/mL) and C 44 Mab-46 (1 µg/mL) for 1 h at room temperature and then treated with the EnVision+ Kit for mouse (Agilent Technologies, Inc.) for 30 min. The color was developed using 3,3 -diaminobenzidine tetrahydrochloride (DAB; Agilent Technologies Inc.) for 2 min. Hematoxylin (FUJIFILM Wako Pure Chemical Corporation) was used for the counterstaining. Leica DMD108 (Leica Microsystems GmbH, Wetzlar, Germany) was used to examine the sections and obtain images.

Institutional Review Board Statement:
The animal study protocol was approved by the Animal Care and Use Committee of Tohoku University (Permit number: 2019NiA-001) for studies involving animals.
Data Availability Statement: All related data and methods are presented in this paper. Additional inquiries should be addressed to the corresponding authors.